Project Summary The ultimate goal of this project is to understand the molecular mechanisms by which RNA processing is controlled by signaling pathways, and the impact of this regulation on cellular function. The specific focus of the current funding period is on regulation of alternative pre-mRNA splicing during T cell activation. Alternative splicing is an essential and ubiquitous mechanism of gene regulation, which allows for the diversification and control of protein expression in distinct cell-types or environmental conditions. In particular, it is well established that signal-induced alternative splicing is pervasive during neuronal activity, nutrient sensing, oncogenesis and immune function. However, a mechanistic view of how signaling pathways control alternative splicing remains lacking. T cell activation provides an excellent a model system for complex cellular responses, in that multiple signaling pathways are triggered downstream of antigen engagement and act, individually and cooperatively, to induce T cell effector functions. Importantly, several hundred genes are known to undergo alternative splicing in response to T cell activation; however, it remains to be determined how these genes are regulated, which genes are co-regulated by overlapping mechanisms/pathways, and what the ultimate functional consequence is of such regulation. This proposal will address these unanswered questions of signal- induced alternative splicing by leveraging recently developed methodologies and systems to determine: (1) the specific signaling pathways that lead to changes in alternative splicing following antigen stimulation of T cells, and the RNA binding proteins (RBPs) that connect each signaling pathway with their respective splicing targets; (2) the molecular mechanisms by which RBPs and/or cell signaling regulate specific transitions in spliceosome assembly to direct isoform expression; (3) the functional impact of alternative splicing on cell signaling through regulating isoform expression of related kinases; and (4) how regulation of alternative splicing is coordinated with other RNA biogenesis events such as transcription and 3' end processing. Together these studies will provide novel insight regarding the interplay of signaling and splicing in shaping cellular function during T cell activation. Since the signaling pathways induced upon T cell activation are also functional in pathways related to cell growth, metabolism and cancer, the insight gained in these studies will be broadly applicable to numerous cellular responses far beyond the immune system. In addition, the studies proposed here will reveal new paradigms regarding the molecular mechanisms by which cells regulate spliceosome assembly, and how RBPs coordinately control splicing along with other steps in RNA processing. Results from these experiments will thus significantly increase understanding of the mechanisms that control alternative splicing, an essential and ubiquitous process for regulating gene expression across all human cell types.